Here we identified two groups of HIV-1 gp120 mutants and used the ligand-binding phenotypes of these mutants to reach the conclusion that the gp120 glycoprotein can assume at least two distinct conformations. This conclusion necessitates that we distinguish among global misfolding, changes in conformational state, and local alterations of epitopes as consequences of the amino acid changes studied. One group of mutants, exemplified by the 375 S/W glycoprotein, bound CD4 and CD4i antibodies well; however, these mutants were inefficiently recognized by CD4BS antibodies. The second group, exemplified by the 423 I/P mutant, bound CD4BS antibodies but not CD4 or CD4i antibodies. The ability of each of these mutants to bind one of these sets of conformation-dependent ligands rules out global misfolding of the altered glycoproteins.
Several pieces of evidence support the explanation that an alteration in gp120 conformational state accounts for the observed phenotype of the 375 S/W mutant. The indole ring of the substituted tryptophan residue in this mutant is expected to fill the Phe 43 cavity and to increase the propensity of gp120 to assume a conformation close to that of the CD4-bound state. The microcalorimetry studies confirmed that both the enthalpic and entropic changes associated with the binding of 375 S/W gp120 and sCD4 are significantly decreased compared with those seen for wild-type gp120 and sCD4. The entropic gains associated with the 375 S/W change more than compensate for the unfavorable enthalpic changes, resulting in a sixfold increase in CD4-binding affinity. Multiple ligands, particularly those interacting with the receptor-binding surfaces of gp120, induce large compensating changes in enthalpy and entropy upon binding gp120 (Kwong et al., submitted). Thus, it is virtually certain that the source of these changes is the gp120 glycoprotein.
Free gp120 is thought to exhibit interdomain flexibility and thus to sample many conformations. Ligands like CD4 that bind across the gp120 domains decrease the entropy of gp120 and promote the formation of energetically favorable interdomain bonds. The tryptophan substitution at residue 375 results in similar changes, strongly suggesting that this substitution favors the sampling by free gp120 of conformations closer to the CD4-bound state. The small but reproducible increase in CCR5 binding of the 375 S/W mutant in the absence of sCD4 is consistent with this model.
Compared to the wild-type gp120 glycoprotein, the 375 S/W mutant was precipitated inefficiently by the CD4BS antibodies. The preferred conformation of the 375 S/W mutant is apparently not suitable for binding the CD4BS antibodies. This interpretation is supported by the limited solvent accessibility in the CD4-bound state of many of the gp120 residues implicated by mutagenesis in the binding of the CD4BS antibodies (39
). A direct effect of the tryptophan substitution on the CD4BS epitopes is not possible if gp120 maintains a CD4-bound conformation, because residue 375 is not solvent accessible in this case (39
). Thus, whether the effect of the tryptophan substitution at serine 375 on the binding of CD4BS antibodies is mediated by conformational alteration or epitope disruption, gp120 must assume different conformations when binding CD4 and CD4BS antibodies.
The argument that CD4 and CD4BS antibodies recognize distinct gp120 conformations is further supported by the phenotypes of the 423 I/P and 423 I/M + 425 N/K + 431 G/E mutants. The substitutions in these mutants were designed to alter the architecture of the β20 and β21 gp120 strands, two critical components of the bridging sheet and CD4-binding region (38
). As expected, these mutants were markedly defective in binding CD4. That the effect of the 423 I/P change on CD4 binding is secondary to conformational disruption is supported by the observations that isoleucine 423 does not contact CD4 (38
) and that a 423 I/S change does not affect CD4 binding (72
Binding of the 423 I/P and 423 I/M + 425 N/K + 431 G/E mutants to the 17b CD4i antibody was also poor, as expected from the contribution of the bridging sheet to contacts with this antibody (38
). In the available X-ray crystal structures, isoleucine 423 directly contacts the 17b Fab fragment, and another change in this gp120 residue, 423 I/S, has previously been shown to eliminate 17b binding (72
). The 423 I/S change also eliminates the binding of another CD4i antibody, 48d, and neither 17b nor 48d binding can be restored by incubation of the mutant gp120 glycoprotein with sCD4 (72
). These observations suggest that the negative effects of the 423 I/P change on 17b and 48d binding probably result from alteration of a side chain that is critical for antibody contact. By contrast, the negative effects of the 423 I/P change on the binding of another subset of CD4i antibodies appear to be mediated through conformational disruption. Three newly described CD4i antibodies (23e, 21c, and 49e) failed to recognize the 423 I/P mutant but, in contrast to the results seen for the 17b and 48d antibodies, precipitated the mutant following incubation with high concentrations of sCD4 (data not shown). Moreover, the 23e, 49e, and 21c antibodies efficiently recognized the 423 I/S mutant even in the absence of sCD4 (98a
Despite the major disruption of the epitopes for both CD4 and CD4i antibodies, the recognition of the 423 I/P and 423 I/M + 425 N/K + 431 G/E mutants by the CD4BS antibodies was efficient and, in the case of the IgG1b12 antibody, even increased relative to that of the wild-type protein. The epitope of the IgG1b12 antibody has been modeled by gp120 mutagenesis and structural analysis of the free antibody (74
). These studies have suggested extensive IgG1b12 contacts with the outer domain of gp120, a model consistent with our results. We recently observed that IgG1b12 binding does not lead to large reductions in gp120 entropy, in contrast to the binding of most CD4BS antibodies (Kwong et al., submitted). An IgG1b12 epitope centered on the outer gp120 domain and not reliant on contacts across gp120 domains would explain this observation. The available data suggest that, although the epitopes of various CD4BS epitopes differ, they are all relatively insensitive to changes that disrupt the conformational integrity of the bridging sheet. In this respect, CD4BS epitopes differ from the binding sites for CD4, CD4i antibodies, and CCR5, which are thought to bind similar or identical conformations of gp120.
The 375 S/W envelope glycoproteins were able to support HIV-1 infection, albeit at a reduced level compared with that of the wild-type envelope glycoproteins. This result suggests that the Phe 43 cavity is not absolutely required for envelope glycoprotein function. Examination of primate immunodeficiency virus sequences reveals that, although most HIV-1 strains have a serine residue at position 375, group O HIV-1 strains generally have a histidine and chimpanzee strains a methionine at this position. A tryptophan residue is found at this position in most HIV-2 and simian immunodeficiency virus (SIV) isolates. One of the Phe 43 cavity-lining residues, tryptophan 112 in HIV-1, is a phenylalanine in HIV-2/SIV gp120 glycoproteins, perhaps allowing tryptophan 375 to be accommodated. The highly conserved nature of the other gp120 residues contacting the Phe 43 cavity (38
) suggests that gp120 architecture in this region is similar among the primate immunodeficiency viruses. Thus, tryptophan 375 in the HIV-2 and SIV gp120 molecules probably represents a cavity-filling residue and might contribute to some of the properties of these viruses that differ from those of HIV-1. For example, SIV strains often exhibit some degree of CD4 independence. HIV-2 and SIV rarely, if ever, elicit CD4BS antibodies, a property that might be explained by preferred gp120 conformations approximating the CD4-bound state and not recognized by CD4BS antibodies.
The 375 S/W change influences the sensitivity of the virus to neutralization. Compared with the wild-type virus, viruses with 375 S/W envelope glycoproteins exhibited a significant increase in sensitivity to the 2G12 antibody, a slight increase in sensitivity to sCD4, and a marked resistance to the CD4BS antibody IgG1b12. These neutralization phenotypes can be explained by the observed alterations in the affinity of monomeric 375 S/W gp120 preparations for 2G12, sCD4, and IgG1b12. The observed alterations in neutralization sensitivity imply that different conformations can be assumed by the gp120 molecule in the context of the wild-type HIV-1 envelope glycoprotein trimer, at least in the presence of particular ligands. More studies will be required to assess the degree of gp120 conformational flexibility on the free envelope glycoprotein trimer.
The conformational flexibility of the HIV-1 envelope glycoproteins is important to the function of these molecules in mediating virus entry and in evading the humoral immune response. Therefore, understanding the range of conformations available to these glycoproteins is important for an appreciation of their role in HIV-1 replication and for guiding attempts at intervention. The feasibility of targeting the conserved receptor-binding regions of HIV-1 gp120 with drugs or antibodies will no doubt be influenced by the conformational variation of these structures. Limiting the conformational heterogeneity of the gp120 core, as we have begun to do with the 375 S/W change, might increase the efficiency with which antibodies directed against receptor-binding surfaces are generated. Although the 375 S/W mutant does not fully mimic the CD4-bound state, the phenotypes observed and the approaches used herein will be useful in guiding efforts to modify the HIV-1 envelope glycoprotein further to achieve that end. Stabilization of other conformational states of the gp120 glycoprotein, such as that recognized by CD4BS antibodies, would also be a desirable goal.